Heterocyclic inhibitors of monocarboxylate transporter

ABSTRACT

The invention provides compounds that inhibit monocarboxylate transporters, such as MCT1 and MCT4. Compounds of the invention can be used for treatment of a condition in a patient, wherein the condition is characterized by the heightened activity or by the high prevalence of MCT1 and/or MCT4, such as cancer or type II diabetes.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. Provisional ApplicationSer. No. 62/106,479, filed Jan. 22, 2015, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R01 CA154739awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

In the 1920s the German biochemist Otto Warburg described metabolicdifferences between cancerous and normal cells, where he noted thattumor cells rely upon a high rate of aerobic glycolysis rather thanoxidative phosphorylation to produce energy for maintenance of cellularfunctions.^(1,2) Indeed, cancer cells have up to a 60-fold enhanced rateof glycolysis relative to normal cells, even with sufficient oxygen.¹This dependence upon glycolysis, and its consequences, is termed “theWarburg effect”.²

Malignant cells are highly anabolic and require very high levelsnutrients, ATP and building blocks to synthesize components needed fortheir growth and survival. Use of the glycolytic pathway provides ATPbut also drives production of lactate, which is produced from pyruvateat the end of the glycolytic pathway. Massive lactate production by thetumor cell requires an efficient means for its consumption orelimination, to prevent intracellular acidification of the cancer cell.

Two mechanisms for handling excess lactate have been described. First,in some rare tumor types lactate is converted to pyruvate for entry intothe TCA cycle. More commonly, however, lactate homeostasis is maintainedvia a family of twelve-membrane pass cell surface proteins known as themonocarboxylate transporters (MCTs; also known as the SLC16a transporterfamily). Fourteen MCTs are known, but only MCT1, MCT2, MCT3 and MCT4transport small monocarboxylates such as lactate, pyruvate and ketonebodies (acetoacetate and β-hydroxybutyrate) across plasma membranes in aproton-linked exchange.³ Expression analyses have established that mostaggressive tumor types express markedly elevated levels of MCT1, MCT4 orboth.⁴ The chaperone protein CD147, which contains immunoglobulin-likedomains, is required for MCT1 and MCT4 cell surface expression and isco-localized with the transporters. MCT1, MCT4 and CD147 are now highpriority targets for cancer therapeutics.⁴

The expression of MCT1 and MCT4 is regulated by two major oncogenictranscription factors, MYC and hypoxia inducible factor-1α (HIF-1α),respectively,^(4,5) that direct marked increases in the production ofkey proteins that support aerobic glycolysis, including amino acidtransporters and enzymes involved in the catabolism of glutamine andglucose.⁶ Malignancies having MYC involvement and hypoxic tumors aregenerally resistant to current frontline therapies, with high rates oftreatment failure, relapse and high patient mortality.^(7,8)Importantly, inhibition of MCT1 or MCT4 can kill tumor cells ex vivo andprovoke tumor regression in vivo,^(4,9) and their potency is augmentedby agents such as metformin that force a glycolytic phenotype upon thecancer cell.⁴

Many weak MCT inhibitors (i.e., those effective at high micromolarlevels) have been described, includingα-cyano-4-hydroxycinnamate^(10,11) stilbene disulfonates,¹² phloretin¹³and related flavonoids.¹⁴ Coumarin-derived covalent MCT inhibitors havealso recently been disclosed,^(15,16) as have pteridinones.¹⁷

The most advanced MCT1 inhibitors are related pyrrolopyrimidine diones,pyrrolopyridazinones, and thienopyrimidine diones,¹⁸⁻²³ including acompound that has advanced into clinical trials for treating some humanmalignancies.^(24,25) These compounds, and to our knowledge all MCT1inhibitors yet described, are dual MCT1/MCT2 inhibitors. MCT2 has veryhigh sequence homology with MCT1, yet it likely has a lesser role thanMCT1 and MCT4 for monocarboxylate transport in human cancers based uponexpression studies. However, MCT2 inhibition may play a role inpotential off-target effects of current agents that could arise fromblocking lactate transport in normal cells.

The first highly potent MCT inhibitor was initially identified via acell-based assay seeking immunosuppressive agents that inhibitNFAT1-directed IL-2 transcription.²⁶ MCT1 inhibition as its mechanism ofaction was described a full decade later.¹⁸ Several subsequentlypublished analogs are also potent MCT1 inhibitors, with low nanomolar Kivalues for MCT1 inhibition and low nanomolar EC₅₀ values inn MTT assaysfor growth of MCT1-expressing tumors.

In many human tumors MCT1 and MCT4 are inversely expressed. Smallmolecule MCT1 inhibitors are now known to disable tumor cell metabolism,proliferation and survival, and impair tumorigenic potential in vivo intumors expressing high levels of MCT1.⁴ MCT4 inhibitors are likely to besimilarly effective for tumors expressing elevated levels of MCT4.Antitumor effects of MCT1 inhibitors are augmented by co-administrationof the biguanide metformin, which is thought to further augment relianceby tumor cells upon aerobic glycolysis and thus increase the demand toMCT1-mediated efflux of lactate.⁴

In addition to antitumor effects, inhibitors of MCT1 and/or MCT4 mayhave other important biological effects, such as immune suppression,¹⁸anti-inflammatory,²⁶ and anti-diabetic effects.²⁷⁻³² MCT1 is normallyexpressed at very low levels in pancreatic islets and in beta-cells inparticular.²⁷⁻²⁸ This scenario explains the very slow uptake of lactatein these cells.²⁹ A hallmark of exercise-induced hyperinsulinism (EIHI)is inappropriate insulin secretion following vigorous physical activity,which leads to hypoglycemia.³⁰ In a 2012 study, Rutter and co-workersestablished that EIHI is associated with elevated expression of MCT1 inbeta-cells and that transgenic mice engineered to overexpress MCT1 inpart displayed many of the hallmarks of EIHI6.³¹ While the link betweenlactate and insulin secretion has been suggested since the late 1980s³²these more recent studies clarify the central role of MCT1 (and perhapsof the related lactate transporters MCT2 and MCT4).

SUMMARY

The present invention provides, in various embodiments, a compound offormula A, B, or C:

wherein

R¹ is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, and (C₁-C₆)fluoroalkyl;

R² is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, (C₁-C₆)fluoroalkyl, a(C₆-C₁₀)aryl ring system, a 5- to 9-membered heteroaryl ring system, a(C₁-C₆)alkyl-(C₆-C₁₀)aryl ring system, and a (C₁-C₆)alkyl-(5- to9-membered)heteroaryl ring system;

provided that when R² comprises an aryl or heteroaryl ring system, thering system bears 0-2 independently selected substituents from the groupconsisting of fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, and(C₁-C₆)fluoroalkoxy;

R³ is a monocyclic or bicyclic (C6-C10) aryl or a monocyclic or bicyclic(5- to 10-membered) heteroaryl group, wherein the aryl or heteroaryl canbe substituted or unsubstituted;

R⁴ is hydrogen, (C₁-C₆)alkyl, (C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl,(C₆-C₁₀)aryl, (5- to 7-membered)heteroaryl, or (4- to 7-membered)saturated heterocyclyl with 1-2 instances of heteroatoms selected fromthe group consisting of NH, NMe, O, and S;

for structure A, Z is CH₂, CH((C₁-C₆)alkyl), CH((C₃-C₇)cycloalkyl), O,N, S, S(O), or SO₂;

for structures B and C, Z is CH₂, CH((C₁-C₆)alkyl),CH((C₃-C₇)cycloalkyl), or O;

n=1, 2, or 3;

the cyclic group indicated as “ring” is an aryl or heteroaryl group ofany one of the following:

wherein the wavy lines indicate points of bonding, and wherein M isindependently selected CH or N, provided that M group can be a nitrogenatom in 0, 1, or 2, instances;

L is S, O, NH, N(C₁-C₆)alkyl, or NCF₃,

each Q is independently CH or N;

wherein R⁵ is optionally present, when present, R⁵ is one to fourinstances of independently selected F, Cl, Br, CF₃, (C₁-C₆)alkyl, OCF₃,O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl; or,

the cyclic group indicated as “ring” is a (C₃-C₇)cycloalkyl or asaturated (3- to 7-membered)heterocyclyl comprising 1-2 heteroatomsselected from the group consisting of O, NH, N(C1-C6)alkyl, andN(C1-C6)fluoroalkyl; wherein the points of bonding may be cis or trans;wherein R⁵ is optionally present, when present, R⁵ is one to fourinstances of independently selected F, Cl, Br, CF₃, (C₁-C₆)alkyl, OCF₃,O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof.

The invention further provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically acceptable excipient.

In various embodiments, the invention provides a method of inhibitingmonocarboxylate transporter MCT1, monocarboxylate transporter MCT4, orboth, comprising contacting the monocarboxylate transporter with aneffective amount or concentration of a compound of the invention.

The invention further provides a method of treatment of a condition in amammal wherein treatment of the condition with a compound having aninhibitor effect on MCT1, MCT4, or both is medically indicated,comprising administering an effective amount of a compound of theinvention. For instance, in various embodiments, a compound of theinvention shows an anti-tumor, anti-diabetes, anti-inflammatory, orimmunosuppressive pharmacological effect. The inventive compounds can beused for treatment of a condition in a patient wherein the condition ischaracterized by the heightened activity or by the high prevalence ofMCT1 and/or MCT4. For instance, the condition can be cancer or type IIdiabetes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structural scaffolds of compounds of the invention.

FIG. 2 shows numbering schemes for the scaffolds.

FIG. 3 shows the chemical structure of the compound of Example 9(SR-11105).

FIG. 4 shows the chemical structure of the compound of Example 21(SR-13779).

FIG. 5 shows a graph indicating the time course of tumor volumereduction caused by administration of the compound of Example 9(SR-11105) relative to vehicle.

FIG. 6 shows a graph indicating the time course of tumor volumereduction caused by administration of the compound of Example 21(SR-13779) relative to vehicle.

FIG. 7 shows a comparison of tumor weight following administration ofthe compounds of Examples 9 (SR-11105) and 21 (SR-13779) relative tovehicle.

FIG. 8 shows a comparison of animal body weight following administrationof the compounds of Examples 9 (SR-11105) and 21 (SR-13779) relative tovehicle.

DETAILED DESCRIPTION

The terms MCT1 and MCT4 refer to monocarboxylate transporter 1 andmonocarboxylate transporter 4, respectively.

The term “inhibitor” as used herein refers to a compound that binds to atarget and renders it biologically inactive or less active.

The term “heteroatom” as used herein refers to an atom of any elementother than carbon or hydrogen. Common heteroatoms include nitrogen,oxygen, phosphorus, sulfur and selenium.

The abbreviation “CNS” as used herein refers to the central nervoussystem of an organism.

The term “EC₅₀” as used herein refers to the dose of a test compoundwhich produces 50% of its maximum response or effect in an assay.

The term “IC₅₀” as used herein refers to the dose of a test compoundwhich produces 50% inhibition in a biochemical assay.

The term “alkyl” as used herein throughout the specification, examples,and claims refers to a hydrocarbon group, and includes branched chainvariations, or “branched alkyl” groups.

The term “cycloalkyl” as used herein throughout the specification,examples, and claims refers to a cyclic hydrocarbon group, and mayinclude alkyl substituents on the cyclic hydrocarbon group.

The term “substituted alkyl” as used herein refers to alkyl moietieshaving substituents replacing a hydrogen atom on one or more carbonatoms of the hydrocarbon backbone. Such substituents can include, forexample, a halogen, a halogenated alkyl (e.g., CF₃), a hydroxyl, acarbonyl, an amino, an amido, an amidine, an imine, an alkoxy, ahalogenated alkoxy (e.g., OCF₃, OCHF₂, etc.) a cyano, a nitro, an azido,a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic group. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate.

The term “aryl” and “heteroaryl” as used herein includes 5-, 6- and7-membered single-ring aromatic groups that may include from zero tofour heteroatoms, for example, benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine, pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls. The terms ortho, meta and para apply to1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example,the names “1,2-dimethylbenzene” and “ortho, meta-dimethylbenzene” aresynonymous.

The term “aralkyl” as used herein refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group). Examplesinclude CH₂Ph, CH₂CH₂Ph, CH₂CH₂-indole, and the like. The aromatic ringcan be substituted at one or more ring positions with such substituents,as described above.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The terms “heterocyclyl” or “heterocyclic group” as used herein refer to3- to 10-membered ring structures, more preferably 3- to 7-memberedrings that include one to four heteroatoms. Heterocycles can also bepolycycles. Heterocyclyl groups include, for example, azetidine,azepine, thiophene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

As used herein, the term “halogen” designates —F, —Cl, —Br or —I.

As used herein, the term “hydroxyl” means —OH.

As used herein, the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” as used herein are recognized in the artand refer to both unsubstituted and substituted amines, e.g., a moietythat can be represented by the general formulas —NH₂, —NHR, —NRR″, whereR and R′ are alkyl, cycloalkyl, aryl, or heterocyclyl groups, asexample.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like.

The term “ether” as used herein refers to two hydrocarbons groupscovalently linked by an oxygen atom.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula —SO₂—N(R)(R′) wherein where R, andR′ are alkyl, cycloalkyl, aryl, or heterocyclyl groups, as examples.

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula —SO₂R wherein where R is an alkyl,cycloalkyl, aryl, or heterocyclyl group, as examples.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. The phrase “protecting group” as usedherein means temporary substituents which protect a potentially reactivefunctional group from undesired chemical transformations. Examples ofsuch protecting groups include carbamates of amines, esters ofcarboxylic acids, silyl ethers of alcohols, and acetals and ketals ofaldehydes and ketones, respectively. The field of protecting groupchemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. ProtectiveGroups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

The term “Example” as used herein indicates the procedures followed forthe preparation of a claimed compound, In general, the compounds of thepresent invention may be prepared by the methods illustrated in thegeneral reaction schemes as, for example, described in the examples, orby modifications thereof, using readily available starting materials,reagents and conventional synthesis procedures not mentioned here.

The invention provides, in various embodiments a compound of formula A,B, or C

wherein:

R¹ is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, and (C₁-C₆)fluoroalkyl;

R² is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, (C₁-C₆)fluoroalkyl, a(C₆-C₁₀)aryl ring system, a 5- to 9-membered heteroaryl ring system, a(C₁-C₆)alkyl-(C₆-C₁₀)aryl ring system, and a (C₁-C₆)alkyl-(5- to9-membered)heteroaryl ring system;

provided that when R² comprises an aryl or heteroaryl ring system, thering system bears 0-2 independently selected substituents from the groupconsisting of fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, and(C₁-C₆)fluoroalkoxy;

R³ is a monocyclic or bicyclic (C6-C10) aryl or a monocyclic or bicyclic(5- to 10-membered) heteroaryl group, wherein the aryl or heteroaryl canbe substituted or unsubstituted;

R⁴ is hydrogen, (C₁-C₆)alkyl, (C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl,(C₆-C₁₀)aryl, (5- to 7-membered)heteroaryl, or (4- to 7-membered)saturated heterocyclyl with 1-2 instances of heteroatoms selected fromthe group consisting of NH, NMe, O, and S;

for structure A, Z is CH₂, CH((C₁-C₆)alkyl), CH((C₃-C₇)cycloalkyl), O,N, S, S(O), or SO₂;

for structures B and C, Z is CH₂, CH((C₁-C₆)alkyl),CH((C₃-C₇)cycloalkyl), or O;

n=1, 2, or 3;

the cyclic group indicated as “ring” is an aryl or heteroaryl group ofany one of the following:

wherein wavy lines indicate points of bonding, and wherein M isindependently selected CH or N, provided that M group can be a nitrogenatom in 0, 1, or 2, instances;

L is S, O, NH, N(C₁-C₆)alkyl, or NCF₃;

each Q is independently CH or N;

wherein R⁵ is optionally present, when present, R⁵ is one to fourinstances of independently selected F, Cl, Br, CF₃, (C₁-C₆)alkyl, OCF₃,O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl; or,

the cyclic group indicated as “ring” is a (C₃-C₇)cycloalkyl or asaturated (3- to 7-membered)heterocyclyl comprising 1-2 heteroatomsselected from the group consisting of O, NH, N(C1-C6)alkyl, andN(C1-C6)fluoroalkyl; wherein the points of bonding may be cis or trans;wherein R⁵ is optionally present, when present, R⁵ is one to fourinstances of independently selected F, Cl, Br, CF₃, (C₁-C₆)alkyl, OCF₃,O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof.

For example, in various embodiments, for a compound of the invention,when the R³ group is monocyclic, the core ring system can consist of 5or 6 atoms in total, with 1-6 carbon atoms, 0-4 nitrogen atoms, 0-2oxygen atoms, and 0-1 sulfur atoms. A few representative examples areshown below:

wherein X is H, (C₁-C₆)alkyl, or CF₃; and

Y is optionally present and, when Y is present, Y is 1-3 instances of asubstituent selected from the group consisting of F, Cl, Br, CF₃,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, NH₂, NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂,NH—(CH₂)_(j)—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding.

When the R³ group is bicyclic, the core ring system can consist of 9 or10 atoms, with 4-10 carbon atoms, 0-6 nitrogen atoms, 0-2 oxygen atoms,and 0-2 sulfur atoms.

Representative examples of 9-atom ring systems are shown below:

wherein the group X is H, (C₁-C₆)alkyl, or CF₃; and

Y is optionally present and, when Y is present, Y is 1-3 instances of asubstituent selected from the group consisting of F, Cl, Br, CF₃,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, NH₂, NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂,NH—(CH₂)₁—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding, and wherein Y can bedisposed on any ring of a multi-ring system.

Representative examples of 10-atom ring systems are shown below:

wherein the group X is H, (C₁-C₆)alkyl, or CF₃; and

Y is optionally present and, when Y is present, Y is 1-3 instances of asubstituent selected from the group consisting of F, Cl, Br, CF₃,(C₁-C₆)alkyl, O(C₁-C₆)alkyl, NH₂, NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂,NH—(CH₂)_(j)—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding, and wherein Y can bedisposed on any ring of a multi-ring system.

EXAMPLES

Chemistry Methods

All reactions were performed in flame-dried glassware fitted with rubbersepta under positive pressure of nitrogen or argon, unless otherwisenoted. Tetrahydrofuran, DMF, acetonitrile, and methylene chloride werepurchased from Aldrich and used as received.

Commercially available reagents were used without further purification.Thin layer chromatography (TLC) analyses were performed on pre-coated250 μM silica 60 F254 glass-backed plates. Flash chromatography wasperformed on pre-packed columns of silica gel (230-400 mesh, 40-63 μm)by CombiFlash with EA/hexane or MeOH/DCM as eluents. Preparative HPLCwas performed on a Shimadzu LC-8A preparative HPLC instrument on SunFireC₁₈ OBD 10 μm (30×250 mm) with CH₃CN+50% MeOH/H₂O+0.1% TFA as eluents topurify the targeted compounds. LC-MS was performed on AgilentTechnologies 1200 series analytical HPLC instrument paired with a 6140quadrupole mass spectrometer or with a Thermo Scientific UltiMate 3000mass spectrometer. Analytical HPLC was performed on Agilent technologies1200 series with CH₃CN (Solvent B)/H₂O+0.9% CH₃CN+0.1% TFA (solvent A)as eluents, and the targeted products were detected by UV in thedetection range of 215-310 nm. ¹H and ¹³C NMR spectra were recorded on aBruker NMR spectrometer at 400 MHz (¹H) or 100 MHz (¹³C). Unlessotherwise specified, CDCl₃ was used as the NMR solvent. Resonances werereported in parts per million downfield from TMS standard, and werereferenced to either the residual solvent peak (typically ¹H: CHCl₃ δ7.27; ¹³C: CDCl₃ δ 77.23).

Certain abbreviations for common chemicals were used in the Examples andare defined as follows:

-   EA=ethyl acetate-   ESI=Electrospray ionization mass spectroscopy-   NMR=nuclear magnetic resonance spectroscopy-   DMSO=dimethyl sulfoxide-   DMF=N,N-dimethylformamide-   Hex=hexanes-   LC-MS=liquid chromatography-mass spectroscopy-   HPLC=high performance liquid chromatography-   NMO=N-methylmorpholine N-oxide-   NMP=N-methyl pyrrolidinone-   TEA=triethylamine-   DIAD=diisopropyl azodicarboxylate-   Tf=trifluoromethansulfonyl    The following compounds are reported as specific examples:

TABLE 1 internal Example chemical structure type ID # groups present 1

A 13219

2

A 14280

3

A 14018

4

A 14020

5

A 13939

6

A 13778

7

A 13218

8

A 14162

9

A 11105

10

A 11057

11

A 11104

12

A 11058

13

A 10345

14

A 10346

15

A 10267

16

A 14163

17

A 14281

18

A 14019

19

A 13938

20

A 13780

21

A 13779

22

A 11241

23

A 11293

24

A 11213

25

B 10366

26

B 10385

27

B 10263

28

B 10154

29

B 10155

30

B 10156

31

B 10049

32

B 10050

33

B 10384

34

B 10383

35

B 10367

36

B 10100

37

B 10603

38

B 10501

39

B 10426

40

B 10344

41

B 10264

42

B 10265

43

B 10318

44

B 10317

45

B 10430

46

B 13990

47

B 11718

48

B 14078

49

B 13991

50

B 13459

51

B 13718

52

B 13758

General Synthesis Scheme for Examples 1-14:

Example 16-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-fluoro-3-(hydroxymethyl)phenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1.

General procedure for the preparation of pinacolbornane reagents: Anoven-dried vial filled with argon was charged with Pd₂dba₃ (2 mol %),X-Phos (4 mol %), bis(pinacolato)diboron (3 equiv.) and KOAc (3 equiv.).1,4-Dioxane was added, followed by the addition of the aryl chloride orbromide (0.5 M in 1,4-dioxane). The vial was sealed, and the reactionmixture was heated to 110° C. until aryl halide had been completelyconsumed, as determined by TLC analysis. At this point the reactionmixture was allowed to cool to room temperature. The reaction solutionwas then filtered through a thin pad of Celite (eluting with ethylacetate) and the eluent was concentrated under reduced pressure. Thecrude material so obtained was purified via flash chromatography onsilica gel or utilized for Step 2m the Suzuki coupling reaction,directly and without purification.

Step 2.

General procedure for the Suzuki reaction: An oven-dried vial filledwith argon was charged with a mixture of the bromothiophene startingmaterial (0.05 M in toluene, synthesis is described below), the boranereagent from step 1 (2 equiv.), Pd(PPh₃)₄ (0.2 equiv.), K₂CO₃ (3equiv.), in minimal toluene-H₂O (3:1, V/V). The vial was sealed and thereaction mixture was heated to 110° C. overnight. After cooling, waterwas added and the reaction mixture was extracted three times with ethylacetate. The combined organic layer was dried over anhydrous Na₂SO₄, andevaporated under reduced pressure. The crude product was purified byflash chromatography on silica gel to yield the product.

Data for the product of example 1: white solid, yield 36% from theSuzuki reaction, ¹H NMR (400 MHz, CD₃OD) δ 7.53 (m, 1H), 7.24 (m, 2H),4.72 (s, 2H), 3.76 (m, 2H), 3.69 (s, 2H), 3.26 (s, 3H), 2.27 (m, 1H),2.06 (s, 6H), 0.96 (d, J=6.56 Hz, 6H). ¹³C NMR (101 MHz, CD₃OD andCDCl₃) δ 160.3, 159.6, 157.8, 153.5, 152.1, 135.8, 131.8, 130.2, 129.8,129.6, 129.2, 124.6, 123.4, 123.3, 58.8, 56.8, 28.4, 28.1, 22.2, 20.2,10.5.

Preparation of the bromide starting material5-bromo-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione:

Step A. A flame dried 50-mL round bottom flask purged with argon wascharged with ethyl 2-amino-5-methylthiophene-3-carboxylate (5 g, 27mmol) and sodium triacetoxyborohydride (8.5 g, 41 mmol). CH₂Cl₂ (80 mL)was added followed by AcOH (1.5 mL, 27 mmol) and the reaction was cooledto 0° C. in an ice/water bath. Isobutyraldehyde (3 mL, 27 mmol) wasadded drop-wise then the mixture was allowed to warm to room temperatureand stir for 16 hours. Reaction was quenched with H₂O and extracted withCH₂Cl₂. Organic layer was dried over MgSO₄, filtered and concentratedunder reduced pressure. The crude material was purified via flashchromatography on silica gel to give the title compound as an off whitesolid (8.3 g, 78%). ¹H NMR (400 MHz, Chloroform-d) δ 7.42 (s, 1H),6.67-6.62 (m, 1H), 4.23 (q, J=7.1 Hz, 2H), 3.01 (t, J=6.4 Hz, 2H), 2.27(s, J=1.3 Hz, 3H), 1.96 (dp, J=13.4, 6.7 Hz, 1H), 1.32 (t, J=7.1 Hz,3H), 0.98 (d, J=6.7 Hz, 6H).

Step B. A flame dried 100-mL 3-neck round bottom flask purged with argonand fitted with a reflux condenser was charged with product from step A(650 mg, 2.7 mmol), triphosgene (269 mg, 0.89 mmol) and toluene (14 mL).Mixture was stirred in a 100° C. oil bath for 3 hours. The mixture wasthen cooled to room temperature and solvent was removed under reducedpressure. 2M methylamine in MeOH (17 mL) was added to the yellow residueand mixture was stirred for 16 hours at room temperature. Mixture wasconcentrated under reduced pressure and purified via flashchromatography on silica gel to give the title compound as a white solid(372 mg, 55%). ¹H NMR (400 MHz, Chloroform-d) δ 6.98 (q, J=1.3 Hz, 1H),3.74 (d, J=7.6 Hz, 2H), 3.41 (s, 3H), 2.44 (d, J=1.3 Hz, 3H), 2.39-2.24(m, 1H), 0.98 (d, J=6.7 Hz, 6H).

Step C. A flame dried 25-mL round bottom flask purged with argon wascharged with product from step B (372 mg, 1.47 mmol). CH₂Cl₂ is addedand the mixture was cooled to 0° C. in an ice/water bath. Br₂ (0.122 mL2.36 mmol) was added drop-wise and mixture was allowed to stir at 0° C.for 1 hour then warmed to room temperature and stirred for an additional4 hours. Br₂ (0.076 mL 1.47 mmol) was added dropwise and mixture wasstirred for 5 hours at room temperature. Reaction was quenched by theaddition of saturated Na₂S₂O₃ aqueous solution, extracted 3 timesCH₂Cl₂. Combined organic layers were dried over MgSO₄, filtered andconcentrated under reduced pressure to yield an orange solid. Crudematerial was purified via flash chromatography on silica gel to yieldthe title compound as a white solid (346 mg, 70%)¹H NMR (400 MHz,Chloroform-d) δ 3.75 (d, J=7.6 Hz, 3H), 3.41 (s, 4H), 2.38 (s, 4H),2.34-2.26 (m, 1H), 0.98 (d, J=6.7 Hz, 9H).

Step D. A flame dried 200-mL round bottom flask purged with argon andfitted with a reflux condenser was charged with product from step C (4.3g, 13 mmol). CCl₄ (45 mL) was added followed by N-bromosuccinimide (2.31g, 13 mmol) and benzoyl peroxide (126 mg, 0.52 mmol). Mixture wasstirred in a 90° C. oil bath for 45 minutes then cooled to roomtemperature. 1M NaOH aqueous solution was added and the layers werepartitioned. The organic layer was dried over MgSO₄, filtered andconcentrated to yield a white solid as the title compound (5 g, 94%). ¹HNMR (400 MHz, Chloroform-d) δ 4.68 (s, 2H), 3.79 (d, J=7.7 Hz, 2H), 3.41(s, 3H), 2.41-2.23 (m, 1H), 1.00 (d, J=6.7 Hz, 6H).

Step E. A flame dried 500-mL round bottom flask purged with argon andfitted with a reflux condenser was charged with the product from step D(5 g, 12.2 mmol) and CHCl₃ (120 mL). Zn(acac)₂ (3.22 g, 12.2 mmol) wasadded in one portion and the mixture was heated in a 76° C. oil bath for15 minutes then cooled to room temperature. Saturated NaHCO₃ aqueoussolution was added and extracted 2 times with CH₂Cl₂. Hydrazinemonohydrate (1.19 mL, 24.6 mmol) is added to the combined organic layersand this mixture was stirred for 16 hours at room temperature. MgSO₄ wasthen added and the mixture was filtered and concentrated under reducedpressure. Crude material was purified via flash chromatography on silicagel followed by recrystallization in 5:1 hexanes:CH₂Cl₂. Title compoundis isolated as a white solid (1.93 g, 37%). ¹H NMR (400 MHz,Chloroform-d) δ 3.84 (s, 2H), 3.67 (d, J=7.7 Hz, 2H), 3.40 (s, 3H),2.27-2.16 (m, 1H), 2.22 (s, 6H), 0.92 (d, J=6.7 Hz, 6H). ¹³C NMR (101MHz, Chloroform-d) δ 157.58, 152.05, 150.60, 131.65, 112.36, 112.02,104.59, 104.26, 55.55, 28.39, 27.01, 22.91, 20.05, 12.53, 11.11.

Example 26-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-fluoro-3-(hydroxymethyl)-5-methylphenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1, and used(3-bromo-2-fluoro-5-methylphenyl)methanol as the halide reagent to givethe pinacol borane(2-fluoro-5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanolin 58% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J=4.96 Hz, 1H), 7.30(dd, J₁=7.08 Hz, J₂=1.96 Hz, 1H), 4.69 (s, 2H), 2.30 (s, 3H), 1.35 (s,12H).

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 70% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.31 (dd, J₁=6.42 Hz,J₂=1.60 Hz, 1H), 7.01 (dd, J₁=6.36 Hz, J₂=1.84 Hz, 1H), 4.67 (s, 2H),3.75 (m, 2H), 3.67 (s, 2H), 3.26 (s, 3H), 2.36 (s, 3H), 2.26 (m, 1H),2.06 (s, 6H), 0.96 (d, J=6.68 Hz, 6H). ¹³C NMR (101 MHz, CD₃OD) δ 159.4,158.2, 155.8, 153.2, 151.9, 143.4, 135.4, 133.9, 131.9, 130.6, 130.5,129.2, 129.0, 128.8, 122.8, 122.6, 114.7, 113.7, 58.8, 58.7, 56.7, 28.4,27.9, 22.1, 20.8, 20.3, 10.6.

Example 36-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methyl-5-(2,4,6-trifluoro-3-(hydroxymethyl)phenyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1, though thestarting bromide was first prepared from bromination and reduction of2,4,6-triflurobenzaldehyde:

2,4,6-trifluorobenzaldehyde (100 mg, 0.625 mmol) was dissolved inconcentrated H₂SO₄ (0.3 mL) and heated to 60° C. To this was addedN-bromosuccinimide (134 mg, 0.75 mmol) in three portions over a periodof 10 min. After being heated for 3 h under argon, the reaction mixturewas poured into ice water. The product was extracted with hexanes,washed with water and brine, and then dried over anhydrous Na2SO4. Theorganic layer was concentrated under reduced pressure to give2,4,6-trifluoro-3-bromobenzaldehyde (149 mg, 100% yield). ¹H NMR (400MHz, CDCl₃) δ 10.25 (s, 1H), 6.89 (dt, J₁=9.12 Hz, J₂=2.04 Hz, 1H). Thisproduct was dissolved in 2 mL of methanol and to this stirring solutionwas added NaBH₄ (29 mg, 0.75 mmol). The reaction was stirred for 1 h atroom temperature. Then the reaction was diluted with ethyl acetate,washed with saturated aqueous NH₄Cl, followed by brine. The organiclayer was dried over anhydrous Na₂SO₄, and evaporated under reducedpressure yielded 2,4,6-trifluoro-3-bromobenzyl alcohol as a white solid(136 mg, 90% yield). This material was used for Suzuki coupling reactionwithout purification. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (dt, J1=9.08 Hz,J2=2.20 Hz, 1H), 4.76 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 161.8, 161.7,161.6, 160.8, 160.7, 160.6, 160.5, 160.1, 160.0, 159.9, 159.4, 159.3,159.2, 159.1, 158.3, 158.2, 158.1, 157.6, 157.5, 157.4, 113.9, 113.8,113.7, 113.6, 113.5, 113.4, 101.3, 101.2, 101.0, 100.7, 93.9, 93.8,93.6, 93.4, 93.3, 52.6.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 27.7% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.56 (m, 1H), 4.73 (s,2H), 3.60 (d, J=7.64 Hz, 2H), 3.26 (s, 2H), 3.01 (s, 3H), 2.22 (m, 1H),2.01 (s, 6H), 0.87 (d, J=6.68 Hz, 6H).

Example 46-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(6-fluoro-5-(hydroxymethyl)pyridin-3-yl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1, though inthis case the hydroxyl group was protected as the t-butyldimethylsilylether. The boronate product was used in Step 2, the Suzuki reaction,without purification or NMR analysis.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the t-butyldimethylsilyl etherderivative of the title compound as a white solid in 50.5% yield. ¹H NMR(400 MHz, CDCl₃) δ 8.06 (brs, 1H), 7.91 (d, J=9.06 Hz, 1H), 4.11 (ABq,J=7.12 Hz, 2H), 3.73 (d, J=7.64 Hz, 2H), 3.68 (s, 2H), 3.31 (s, 3H),2.25 (m, 1H), 2.11 (s, 6H), 0.96 (d, J=6.68 Hz, 6H), 0.92 (s, 9H), 0.12(s, 6H). To a solution of this silyl ether (12 mg, 0.020 mmol) in 0.5 mLof dry THF was added dropwise via syringe tetra-butylammonium fluoride(0.06 ml). The mixture was allowed to stir at room temperature for 1.5 hbefore addition of saturated Na₂CO₃ (1 mL). The aqueous layer wasextracted with ethyl acetate (2 mL×3). The organic layers were combined,dried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography to give a white solid (2mg, 21% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.05 (brs, 1H), 7.83 (d,J=9.08 Hz, 1H), 4.77 (s, 2H), 3.75 (d, J=7.68 Hz, 2H), 3.70 (s, 2H),3.31 (s, 3H), 2.28 (m, 1H), 2.07 (s, 6H), 0.98 (d, J=6.68 Hz, 6H). ¹³CNMR (101 MHz, CDCl₃) δ 161.1, 159.8, 158.5, 152.5, 150.8, 146.2, 146.0,143.0, 140.8, 133.9, 130.4, 128.9, 122.4, 122.3, 113.5, 110.0, 58.5,56.1, 29.8, 28.3, 27.1, 20.1, 11.0.

Example 55-(3,4-difluoro-5-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 3 step 1.2,3-difluorobenzaldehyde (200 mg, 1.408 mmol) was dissolved inconcentrated H₂SO₄ (0.64 mL) and heated to 60° C. To this was addedN-bromosuccinimide (301 mg, 1.690 mmol) in three portions over a periodof 20 min. After being heated for 3 h under argon, the reaction mixturewas poured into ice water. The product was extracted with hexanes,washed with water and brine, and then dried over anhydrous Na₂SO₄.Purification by flash chromatography yielded an orange liquid as2,3-difluoro-5-bromobenzaldehyde (155 mg, 50% yield), which wasdissolved in 6 mL of methanol. To this stirring solution was added NaBH₄(32 mg, 0.84 mmol). The reaction was stirred for 1 h at roomtemperature. Then the reaction was diluted with ethyl acetate, washedwith saturated aqueous NH₄Cl, followed by brine. The organic layer wasdried over anhydrous Na₂SO₄, and evaporated under reduced pressureyielded 2,3-difluoro-5-bromobenzyl alcohol as a white solid (163 mg, 87%yield). The boronate product was then obtained from2,3-difluoro-5-bromobenzyl alcohol according to the general procedure ofExample 1. The crude material was used for Suzuki coupling reactionwithout purification.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 27.7% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.56 (m, 1H), 4.73 (s,2H), 3.60 (d, J=7.64 Hz, 2H), 3.26 (s, 2H), 3.01 (s, 3H), 2.22 (m, 1H),2.01 (s, 6H), 0.87 (d, J=6.68 Hz, 6H).

Example 65-(2,4-difluoro-5-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1 using5-chloro-2,4-difluorobenzyl alcohol. Purification by flashchromatography gave the boronate as a white solid in 52% yield. ¹H NMR(400 MHz, CDCl₃) δ 7.78 (dd, J₁=9.04 Hz, J₂=6.84 Hz, 1H), 6.87 (t,J=9.44 Hz, 1H), 4.67 (s, 2H), 1.33 (s, 12H). ¹³C NMR (101 MHz, CDCl₃) δ168.7, 168.6, 166.2, 166.0, 164.6, 164.5, 162.1, 162.0, 138.0, 137.9,137.8, 123.8, 123.7, 123.6, 104.0, 103.7, 103.4, 84.1, 58.9, 24.9.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 46.2% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.31 (t, J=8.08 Hz, 1H),6.90 (t, J=9.48 Hz, 1H), 4.73 (ABq, J=13.32 Hz, 2H), 3.73 (m, 2H), 3.64(s, 2H), 3.31 (s, 3H), 2.27 (m, 1H), 2.08 (s, 6H), 0.97 (d, J=6.68 Hz,6H). ¹³C NMR (101 MHz, CDCl₃) δ 160.7, 159.1, 158.4, 152.1, 150.9,143.0, 134.1, 132.1, 131.8, 128.7, 127.4, 124.1, 119.0, 113.7, 113.2,58.7, 56.0, 28.2, 27.1, 21.7, 20.1, 10.8.

Example 75-(2,4-difluoro-3-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1 using3-chloro-2,6-difluorobenzyl alcohol according to the general procedure.Purification by flash chromatography gave the title compound as a whitesolid in 57% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (q, J=7.16 Hz, 1H),6.87 (t, J=8.60 Hz, 1H), 4.76 (s, 2H), 1.34 (s, 12H). ¹³C NMR (101 MHz,CDCl₃) δ 167.5, 167.4, 165.2, 165.1, 165.0, 164.9, 162.7, 162.6, 137.4,137.3, 137.2, 116.2, 116.1, 116.0, 115.8, 111.5, 111.3, 111.2, 84.1,52.9, 24.8.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 43% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.32 (q, J=8.08 Hz, 1H),7.05 (t, J=8.76 Hz, 1H), 4.72 (s, 2H), 3.76 (d, J=7.20 Hz, 2H), 3.71 (d,J=3.36 Hz, 2H), 3.26 (s, 3H), 2.07 (s, 6H), 2.26 (m, 1H), 0.95 (d,J=6.68 Hz, 6H). ¹³C NMR (101 MHz, CD₃OD) δ164.2, 164.1, 161.7, 161.6,159.7, 159.1, 159.0, 153.6, 152.2, 143.8, 136.3, 133.1, 133.0, 128.4,120.0, 119.8, 117.7, 114.6, 113.9, 111.9, 111.7, 56.8, 52.6, 28.4, 28.2,22.2, 20.2, 10.5.

Example 86-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methyl-5-(5-(2,2,2-trifluoro-1-hydroxyethyl)pyridin-3-yl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 4 step 1, with ahydroxyl group protected as the t-butyldimethylsilyl ether. The crudematerial was then used for the Suzuki coupling reaction withoutpurification.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the t-butyldimethylsilyl etherderivative of the title compound as a white solid in 40% yield. ¹H NMR(400 MHz, CDCl₃) δ 8.66 (s, 1H), 8.62 (d, J=1.72 Hz, 1H), 7.83 (s, 1H),5.06 (q, J=6.28 Hz, 1H), 3.73 (d, J=7.68 Hz, 2H), 3.68 (s, 2H), 3.31 (s,3H), 2.27 (m, 1H), 2.16 (s, 6H), 0.96 (d, J=6.60 Hz, 6H), 0.90 (s, 9H),0.16 (s, 3H), 0.05 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.0, 152.5,151.1, 150.9, 147.9, 142.7, 137.0, 134.4, 130.8, 130.5, 130.4, 125.5,113.3, 113.2, 56.0, 53.5, 31.0, 28.2, 27.1, 25.6, 20.1, 18.2, 10.9,−4.8, −5.2. To a solution of this material (18 mg, 0.028 mmol) in 0.5 mLof dry THF was added dropwise via syringe tetra-butylammonium fluoride(0.06 ml). The mixture was allowed to stir at room temperature for 1.5 hbefore addition of saturated Na₂CO₃ (1 mL). The aqueous layer wasextracted with ethyl acetate (2 mL×3). The organic layers were combined,dried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography to give the title compoundas a white solid (6 mg, 40% yield).¹H NMR (400 MHz, CDCl₃) δ 8.66 (brs,1H), 8.56 (d, J=1.88 Hz, 1H), 7.98 (brs, 1H), 5.27 (m, 1H), 3.79 (d,J=7.64 Hz, 2H), 3.76 (d, J=7.64 Hz, 2H), 3.76 (s, 2H), 3.27 (s, 3H),2.27 (m, 1H), 2.05 (s, 6H), 0.96 (d, J=6.68 Hz, 6H). ¹³C NMR (101 MHz,CD₃OD) δ 159.8, 154.1, 152.2, 151.5, 148.2, 138.7, 136.4, 132.9, 132.4,131.4, 130.2, 124.5, 114.2, 114.0, 75.8, 56.9, 28.4, 28.2, 25.0, 22.0,10.5.

Example 96-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(4-fluoro-3-(hydroxymethyl)phenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1 using3-chloro-2-fluorobenzyl alcohol according to the general procedure. Thecrude material was used for Suzuki coupling reaction withoutpurification.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 48% yield. LC-MS (ESI): m/z 471.2 [M+1]⁺.

Example 106-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-(hydroxymethyl)pyridin-4-yl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 4 step 1 thecorresponding des-fluoro starting material according to the generalprocedure. The crude material was used for Suzuki coupling reactionwithout purification.

Step 2 followed the general procedure from Example 9 step 2. ¹H NMR (400MHz, Chloroform-d) δ 8.63 (d, J=5.0 Hz, 1H), 7.29-7.24 (m, 3H), 7.19(dd, J=5.1, 1.6 Hz, 1H), 4.82 (s, 2H), 3.74 (d, J=7.7 Hz, 2H), 3.66 (s,2H), 3.48 (s, 2H), 3.32 (s, 3H), 2.28 (dt, J=13.8, 6.9 Hz, 1H), 2.09 (s,6H), 0.97 (d, J=6.7 Hz, 6H); LC-MS (ESI): m/z 454 [M+1]⁺.

Example 116-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(3-fluoro-5-(hydroxymethyl)phenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 9 step 1 using3-chloro-5-fluorobenzyl alcohol as the starting material according tothe general procedure. The crude material was used for Suzuki couplingreaction without purification.

Step 2 followed the general procedure from Example 9 step 2. LC-MS(ESI): m/z 471 [M+1]⁺.

Example 126-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(5-(hydroxymethyl)pyridin-3-yl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1 using(5-bromopyridin-3-yl)methanol as the starting material according to thegeneral procedure. The crude material was used for Suzuki couplingreaction without purification.

Step 2 followed the general procedure from Example 9 step 2. LC-MS(ESI): m/z 454 [M+1]⁺.

Example 136-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(3-(1-hydroxyethyl)phenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1.3-bromoacetophenone was first reduced to the alcohol using NaBH₄. Thisbromide was the starting material for boronate formation according tothe general procedure. The crude material was then used for the Suzukicoupling reaction without purification.

Step 2 followed the general procedure from Example 9 step 2, LC-MS(ESI): m/z 467 [M+1]⁺.

Example 146-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methyl-5-(3-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1.1-(3-bromophenyl)-2,2,2-trifluoroethanol was the starting material forboronate formation according to the general procedure. The crudematerial was then used for the Suzuki coupling reaction withoutpurification.

Step 2 followed the general procedure from Example 9 step 2. LC-MS(ESI): m/z 521 [M+1]⁺.

Example 156-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(3-(hydroxymethyl)phenyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1 using(3-bromophenyl)methanol as the starting material according to thegeneral procedure. The crude material was used for Suzuki couplingreaction without purification.

Step 2 followed the general procedure from Example 1 step 2. LC-MS(ESI): m/z 453.2 [M+1]⁺.

General Synthesis Scheme for Examples 16-24:

Example 163-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(4-fluoro-3-(hydroxymethyl)phenyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1: (4-fluoro-3-formylphenyl)boronic acid was commercially availableand was used in Step 2.

Step 2 followed the general Suzuki reaction procedure, but using theformylated boronic ester as shown, and followed by reduction:

4-fluoro-3-formylphenylboronic acid and the indicated bromothiophene(synthesis shown below) were used in the general procedure for Suzukicoupling, as in Example 1 Step 2. A mixture of the crude product fromthe Suzuki coupling and NaBH₄ (2 equiv.) was stirred in MeOH for 1 h atroom temperature. The reaction mixture was diluted with ethyl acetate,washed with saturated aqueous NH₄Cl and brine. The organic layer wasdried over anhydrous Na₂SO₄, and evaporated under reduced pressure. Thecrude product was purified by flash chromatography to yield the titlecompound as a white solid in 65.7% yield for two steps. ¹H NMR (400 MHz,CDCl₃) δ 7.38 (dd, J₁=7.07 Hz, J₂=1.64 Hz, 1H), 7.19 (m, 1H), 7.05 (t,J=8.84 Hz, 1H), 4.75 (s, 2H), 3.70 (d, J=7.60 Hz, 2H), 3.59 (s, 2H),2.60 (m, 1H), 2.24 (m, 1H), 2.03 (s, 6H), 1.06 (m, 2H), 0.95 (d, J=6.68Hz, 6H), 0.72 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 161.2, 159.5, 158.7,152.5, 151.6, 142.6, 134.2, 132.7, 130.7, 130.5, 130.4, 130.3, 128.2,128.1, 115.0, 114.7, 113.8, 113.2, 58.8, 55.1, 27.1, 25.3, 20.1, 10.9,9.0.

Preparation of the bromide starting material5-bromo-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione:

Preparation of this bromide followed the procedure used in Example 1 forthe N-Me analog, using cyclopropylamine in place of methylamine in StepB and adding a base treatment step at the end of Step B to promotecyclization. The Step B procedure: A flame dried flask purged with argonand fitted with a reflux condenser was charged with product from step Aof Example 1 (100 mg, 0.41 mmol) and toluene (1.6 mL). Triphosgene (62mg, 0.20 mmol) is added and the mixture was stirred while heating in a100° C. oil bath for 2.5 hours. Cyclopropyl amine (63 μL, 0.91 mmol) wasthen added and mixture was stirred at 100° C. for an additional 2.5hours. Mixture was cooled to room temperature and quenched with water,extracted with ethyl acetate and concentrated under reduced pressure.Crude material was purified via flash chromatography on silica gel toafford the urea compound (76 mg, 57%). ¹H NMR (400 MHz, Chloroform-d) δ8.12 (s, 1H), 6.30 (q, J=1.3 Hz, 1H), 5.57 (s, 1H), 2.97 (d, J=6.7 Hz,2H), 2.27 (d, J=1.2 Hz, 3H), 1.94 (dp, J=13.4, 6.7 Hz, 1H), 0.98 (d,J=6.7 Hz, 6H), 0.85-0.73 (m, 2H), 0.58-0.49 (m, 2H). A flame dried roundbottom flask purged with argon was charged with the product above (71mg, 0.22 mmol), sodium methoxide (47 mg, 0.88 mmol) and methanol (0.8mL). The mixture was stirred for 3 hours at room temperature thenquenched with water, extracted with methoxy ethane, and the combinedorganics were concentrated under reduced pressure. The crude materialwas purified via flash chromatography on silica gel to yield thecyclized product (57 mg, 93%). ¹H NMR (400 MHz, Chloroform-d) δ 6.94 (q,J=1.2 Hz, 1H), 3.71 (d, J=7.6 Hz, 2H), 2.73 (tt, J=7.1, 4.0 Hz, 1H),2.42 (d, J=1.3 Hz, 3H), 2.37-2.21 (m, 1H), 1.24-1.10 (m, 2H), 0.97 (d,J=6.7 Hz, 6H), 0.85-0.75 (m, 2H).

Step C: A flame dried round bottom flask purged with argon was chargedwith product from step 2b (200 mg, 0.72 mmol) and CH₂Cl₂ (2 mL). Bromine(70 μL, 1.08 mmol) was added at room temperature and the mixture wasstirred for 16 hours. Reaction was quenched by the addition of saturatedNa₂S₂O₃ aqueous solution, extracted 3 times CH₂Cl₂. Combined organiclayers were dried over MgSO₄, filtered and concentrated to yield anorange solid. Crude material was purified via flash chromatography onsilica gel to yield the title compound as a white solid (220 mg, 86%).¹H NMR (400 MHz, Chloroform-d) δ 3.72 (d, J=7.6 Hz, 2H), 2.72 (tt,J=7.1, 4.1 Hz, 1H), 2.36 (s, 3H), 2.27 (p, J=7.0 Hz, 1H), 1.28-1.09 (m,2H), 0.97 (d, J=6.7 Hz, 6H), 0.89-0.76 (m, 2H).

Step D followed Step D in example 1 for the N-Me compound. Step Efollowed Step E in example 1 for the N-Me compound, giving thebrominated product: ¹H NMR (400 MHz, Chloroform-f) δ 3.83 (s, 2H), 3.31(s, 7H), 2.72 (tt, J=7.1, 4.1 Hz, 1H), 2.23 (s, 6H), 2.24-2.11 (m, 1H),1.27-1.13 (m, 2H), 0.92 (d, J=6.7 Hz, 6H), 0.85-0.72 (m, 2H).

Example 173-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-fluoro-3-(hydroxymethyl)-5-methylphenyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 2, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 21.4% yield. ¹H NMR (400MHz, CD₃OD) δ 7.32 (dd, J₁=6.40 Hz, J₂=1.60 Hz, 1H), 7.03 (J₁=6.40 Hz,J₂=1.80 Hz, 1H), 4.87 (d, J=2.24 Hz, 2H), 3.75 (m, 2H), 3.68 (brs, 2H),2.57 (m, 1H), 2.37 (s, 3H), 2.23 (m, 1H), 2.04 (s, 6H), 1.03 (m, 2H),0.95 (dd, J₁=6.68 Hz, J₂=1.60 Hz, 6H), 0.68 (m, 2H). ¹³C NMR (101 MHz,CD₃OD) δ 160.7, 158.5, 156.1, 153.7, 153.1, 143.5, 135.5, 134.2, 132.2,130.7, 129.5, 129.3, 129.2, 123.2, 123.0, 115.2, 114.1, 58.9, 56.7,28.3, 26.1, 22.2, 20.8, 20.2, 10.5, 9.6, 9.5.

Example 183-cyclopropyl-5-(3,4-difluoro-5-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 5, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 35% yield. ¹H NMR (400 MHz,CDCl₃) δ 7.18 (d, J=5.12 Hz, 1H), 7.02 (m, 1H), 4.75 (s, 2H), 3.70 (d,J=7.64 Hz, 2H), 3.59 (s, 2H), 2.60 (m, 1H), 2.24 (m, 1H), 2.04 (s, 6H),1.06 (m, 1H), 0.94 (d, J=6.68 Hz, 6H), 0.71 (m, 1H). ¹³C NMR (101 MHz,CD₃OD and CDCl₃) δ 160.2, 153.3, 152.4, 151.6, 149.6, 149.1, 147.1,143.3, 134.8, 133.5, 131.6, 131.5, 131.1, 131.0, 125.9, 118.4, 118.2,114.3, 113.4, 58.2, 56.4, 27.7, 25.7, 22.0, 20.3, 10.7, 9.3.

Example 193-cyclopropyl-5-(2,4-difluoro-5-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 6, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 19% yield. ¹H NMR (400 MHz,CDCl₃) δ 7.31 (t, J=8.16 Hz, 1H), 6.88 (t, J=9.48 Hz, 1H), 4.72 (ABq,J=13.28 Hz, 2H), 3.71 (m, 2H), 3.62 (s, 2H), 2.62 (m, 1H), 2.24 (m, 1H),2.07 (s, 6H), 1.08 (m, 1H), 0.96 (d, J=6.68 Hz, 6H), 0.72 (m, 2H). ¹³CNMR (101 MHz, CDCl₃) δ 159.3, 159.1, 158.4, 152.3, 151.7, 142.9, 133.9,131.7, 127.4, 124.0, 118.5, 114.0, 113.2, 103.9, 103.7, 58.6, 55.8,27.1, 25.3, 21.7, 20.2, 10.8, 8.0.

Example 203-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-fluoro-3-(hydroxymethyl)phenyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 1, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 33% yield: ¹H NMR (400 MHz,CD₃OD) δ 7.52 (m, 1H), 7.23 (m, 2H), 4.72 (s, 2H), 3.73 (m, 2H), 3.66(s, 2H), 2.57 (m, 1H), 2.24 (m, 1H), 2.05 (s, 6H), 1.05 (m, 2H), 0.95(d, J=6.68 Hz, 6H), 0.69 (m, 2H). ¹³C NMR (101 MHz, CD₃OD and CDCl₃) δ160.5, 160.1, 157.6, 153.4, 152.9, 135.6, 131.7, 130.0, 129.6, 129.4,129.1, 124.4, 123.2, 123.1, 115.0, 113.7, 58.8, 58.7, 56.6, 28.0, 25.9,22.1, 20.3, 10.6, 9.5, 9.4.

Example 213-cyclopropyl-5-(2,4-difluoro-3-(hydroxymethyl)phenyl)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 7, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 29% yield: LC-MS (ESI): m/z515.3 [M+1]⁺.

Example 223-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-fluoro-5-(hydroxymethyl)phenyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 followed the general procedure from Example 1 step 1, and used(3-bromo-4-fluorophenyl)methanol as the halide reagent.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 31% yield: LC-MS (ESI): m/z497.2 [M+1]⁺.

Example 233-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(2-(hydroxymethyl)pyridin-4-yl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 10, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 37% yield: ¹H NMR (400 MHz,Chloroform-d) δ 8.61 (dd, J=5.1, 0.8 Hz, 1H), 7.25 (dd, J=1.7, 0.9 Hz,1H), 7.17 (dd, J=5.1, 1.6 Hz, 1H), 5.29 (s, 1H), 4.81 (s, 2H), 3.71 (d,J=7.6 Hz, 2H), 3.63 (s, 2H), 3.48 (s, 2H), 2.63 (tt, J=7.1, 4.0 Hz, 1H),2.32-2.17 (m, 1H), 2.08 (s, 6H), 1.15-1.05 (m, 2H), 0.96 (d, J=6.7 Hz,6H), 0.78-0.68 (m, 2H).

Example 243-cyclopropyl-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(3-(hydroxymethyl)phenyl)-1-isobutylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Step 1 was previously shown as Example 15, Step 1.

Step 2 followed the general procedure from Example 1 step 2 using theN-cyclopropyl-containing bromide. Purification by flash chromatographygave the title compound as a white solid in 24% yield: LC-MS (ESI): m/z479.1 [M+1]⁺.

General Synthesis Scheme for Examples 25-40:

Example 257-(5-(hydroxymethyl)thiazol-2-yl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 1 Step 1 using(2-bromothiazol-5-yl)methanol. The boronate was taken to the next stepwithout purification.

Preparation of the Iodine-Containing Starting Material for Step 2:

LDA (0.5 M, 0.6 mL, 0.3 mmol), was added to a solution of the previousproduct (100 mg, 0.29 mmol) in THF at −78° C. The reaction mixture wasstirred for 30 min. at the same temperature followed by addition of asolution of iodine (74 mg, 0.29 mmol) solution in THF. The reactionmixture was allowed to stir for additional 20 min at −78° C. then warmedup to room temperature. After 2h, the reaction was quenched by sat. aq.NH₄Cl solution and diluted with ethyl acetate. The aqueous phase wasextracted with ethyl acetate (10 mL×2) and the combined organic layerswere washed with brine, dried over. Na₂SO₄, filtered and concentrated.The crude product was purified by flash chromatography to afford7-iodo-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one(81 mg, 59%) as a off white solid. LCMS 472.2 (M+H)⁺.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 43% yield: ¹H NMR (CDCl₃, 400 MHz) 7.94-7.88 (m, 3H), 7.68 (s,1H), 7.57-7.52 (m, 2H), 7.42-7.39 (m, 1H), 7.02 (s, 1H), 6.98-6.96 (m,1H), 6.35 (s, 1H), 4.90 (s, 2H), 3.77 (s, 3H), 2.48 (d, 2H, J=7.2),2.08-1.98 (m, 1H), 0.90 (d, 6H, J=6.4). LCMS 459.2 (M+H)⁺.

Example 267-(2-(hydroxymethyl)thiazol-4-yl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 1 Step 1 using(4-bromothiazol-2-yl)methanol. The boronate was taken to the next stepwithout purification.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25. Purification by flash chromatography gave thetitle compound as a white solid in 41% yield: LCMS 459.2 (M+H)⁺.

Example 277-(2,4-difluoro-3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: see Example 7.

Step 2 followed the general procedure from Example 1 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 36% yield: LCMS 488.2 (M+H)⁺.

Example 287-(2-fluoro-5-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: see Example 22.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25. Purification by flash chromatography gave thetitle compound as a white solid in 46% yield: LCMS 470.2 (M+H)⁺.

Example 297-(2-fluoro-3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: see Example 1.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25. Purification by flash chromatography gave thetitle compound as a white solid in 33% yield: LCMS 470.2 (M+H)⁺.

Example 307-(2-chloro-5-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial (2-chloro-5-(methoxycarbonyl)phenyl)boronic acid wasobtained and used in the Suzuki reaction.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25 to give the methyl ester analog of the desiredproduct, methyl4-chloro-3-(4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-1-oxo-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-7-yl)benzoate.This material was dissolved in minimal dry THF at 0° C. and 2.5 equiv.of LiAlH₄ was added. Standard water/base/water work-up and filtrationgave the crude product. Purification by flash chromatography gave thetitle compound as a white solid in 17% yield for the three steps: LCMS486.2 (M+H)⁺.

Example 317-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial (3-(hydroxymethyl)phenyl)boronic acid was used.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25. Purification by flash chromatography gave thetitle compound as a white solid in 29% yield: LCMS 452.2 (M+H)⁺.

Example 327-(5-(hydroxymethyl)pyridin-3-yl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 1 Step 1 using(5-bromopyridin-3-yl)methanol. The boronate was taken to the next stepwithout purification.

Step 2 followed the general procedure from Example 1 step 2 using theiodide from Example 25. Purification by flash chromatography gave thetitle compound as a white solid in 40% yield: LCMS 453.2 (M+H)⁺.

Example 337-(5-(1-hydroxyethyl)thiophen-3-yl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial 1-(4-bromothiophen-2-yl)ethanone was obtained andused in the Suzuki reaction.

Step 2 followed the general procedure from Example 30 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 14% yield: ¹H NMR (CDCl₃, 400 MHz) 7.86-7.85 (m, 1H), 7.79-7.77(m, 1H), 7.67-7.64 (m, 1H), 7.45-7.42 (m, 2H), 7.38-7.30 (m, 1H),7.16-7.15 (m, 1H), 7.10-7.09 (m, 1H), 6.93 (s, 1H), 6.80-6.78 (m, 1H),5.67 (s, 2H), 5.02-4.97 (m, 1H), 3.63 (s, 3H), 2.44 (d, 2H, J=7.6),2.05-2.02 (m, 1H), 1.44 (d, 3H, J=6.4), 0.85 (d, 6H, J=6.8). LCMS 472.1(M+H)⁺.

Example 347-(6-(1-hydroxyethyl)pyridin-2-yl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial 1-(6-bromopyridin-2-yl)ethanone was obtained and usedin the Suzuki reaction.

Step 2 followed the general procedure from Example 30 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 11% yield: ¹H NMR (CDCl₃, 400 MHz) 7.85-7.72 (m, 5H), 7.46-7.42(m, 2H), 7.37-7.33 (m, 1H), 7.30-7.28 (m, 1H), 7.07 (s, 1H), 6.86-6.84(m, 1H), 6.86 (s, 2H), 4.85-4.78 (m, 2H), 3.68 (s, 3H), 2.47 (d, 2H,J=7.6), 2.06-1.95 (m, 1H), 1.31 (d, 3H, J=6.4), 0.85 (d, 6H, J=6.8).LCMS 467.3 (M+H)⁺.

Example 357-(2-fluoro-5-(1-hydroxyethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial 1-(3-bromo-4-fluorophenyl)ethanone was obtained andused in the Suzuki reaction.

Step 2 followed the general procedure from Example 30 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 16% yield: ¹H NMR (CDCl₃, 400 MHz) 7.90-7.85 (m, 2H), 7.64-7.62(m, 1H), 7.54-7.41 (m, 5H), 7.20-7.07 (m, 2H), 6.99-6.98 (m, 1H),5.67-5.60 (m, 2H), 4.88-4.83 (m, 1H), 3.69 (s, 3H), 2.50 (d, 2H, J=7.6),2.10-2.06 (m, 1H), 1.40 (d, 3H, J=6.4), 0.95-0.91 (m, 6H). LCMS 484.1(M+H)⁺.

Example 367-(3-(1-hydroxyethyl)phenyl)-4-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Commercial1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethanone wasobtained and used in the Suzuki reaction.

Step 2 followed the general procedure from Example 30 step 2.Purification by flash chromatography gave the title compound as a whitesolid in 34% yield: LCMS 466.3 (M+H)⁺.

Example 374-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-7-(2-(2,2,2-trifluoro-1-hydroxyethyl)thiazol-4-yl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1:

To a stirred solution of commercial 4-bromothiazole-2-carbaldehyde (1eq), CF₃TMS (1.1 eq) in dimethoxyethane was added CsF (10 mol %) underargon atmosphere. The reaction mixture was allowed to stir forovernight. The solvent was removed under vacuum, water was added, andthe solution was extracted with ethyl acetate. The combined organiclayers were washed with brine, dried over sodium sulphate, concentratedunder vacuum, and purified by flash chromatography using 10-20% of ethylacetate in hexanes, and isolated in 67% yield. This material was thenconverted to the boronic ester to be used in the Suzuki reaction,following the procedure of Example 1, step 2.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 using the iodide from Example 25 to give the title product,which was purified by flash chromatography and isolated in 45% yield asa white solid: ¹H NMR (CDCl₃, 400 MHz) 8.54 (s, 1H), 7.83-7.73 (m, 3H),7.47-7.7.42 (m, 2H), 7.30-7.26 (m, 1H), 6.93 (s, 1H), 6.79-6.78 (m, 1H),6.03 (s, 2H), 4.99-4.95 (m, 1H), 3.66 (s, 3H), 2.43 (d, 2H, J=7.6),2.04-1.94 (m, 1H), 0.85-0.83 (m, 6H). LCMS 527.1 (M+H)⁺.

Example 384-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-7-(5-(2,2,2-trifluoro-1-hydroxyethyl)thiophen-3-yl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 37 Step 1 using4-bromothiophene-2-carbaldehyde as the starting reagent and followed byconversion to the boronate which was taken to the next step withoutpurification.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 using the iodide from Example 25 to give the title product,which was purified by flash chromatography and isolated in 33% yield asa white solid: LCMS 526.3 (M+H)⁺.

Example 394-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-7-(5-(2,2,2-trifluoro-1-hydroxyethyl)thiophen-2-yl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 37 Step 1 using5-bromothiophene-2-carbaldehyde as the starting reagent and followed byconversion to the boronate which was taken to the next step withoutpurification.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 using the iodide from Example 25 to give the title product,which was purified by flash chromatography and isolated in 36% yield asa white solid: ¹H NMR (CDCl₃, 400 MHz) 7.95-7.94 (m, 1H), 7.93-7.88 (m,1H), 7.75-7.72 (m, 1H), 7.58-7.52 (m, 2H), 7.47-7.43 (m, 1H), 7.12-7.07(m, 2H), 7.03 (s, 1H), 6.97-6.95 (m, 1H), 5.76 (s, 2H), 5.17-5.12 (m,2H), 3.73 (s, 3H), 2.52 (d, 2H, J=7.6), 2.14-2.02 (m, 1H), 0.94 (d, 6H,J=6.8). LCMS 526.3 (M+H)⁺.

Example 404-isobutyl-2-methyl-6-(naphthalen-1-ylmethyl)-7-(3-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 followed the general procedure of Example 37 Step 1 using3-bromobenzaldehyde as the starting reagent and followed by conversionto the boronate which was taken to the next step without purification.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 using the iodide from Example 25 to give the title product,which was purified by flash chromatography and isolated in 32% yield asa white solid: LCMS 520.3 (M+H)⁺.

General Synthesis Scheme for Examples 41-42:

Example 416-([1,1′-biphenyl]-4-ylmethyl)-7-(2-fluoro-5-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: see Example 23.

Preparation of the iodo starting material for Step 2:

To a solution of4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one (50 mg,0.243 mmol) in DMF (10 mL) was added K₂CO₃ (67 mg, 0.49 mmol) at roomtemperature under nitrogen. After 5 min p-bromobenzyl bromide (1.2 eq)was added, The reaction mixture was stirred for an additional 2h. Thesolid was filtered off, DMF was removed under vacuum, the residue wasdissolved in ethyl acetate and was washed with water (20 mL) and brine(20 mL×2). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated. Purification by flash chromatography(20% EtOAc/Hexanes) afforded the intermediate bromide6-(4-bromobenzyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one(67%) as a white solid. LCMS m/z 374 & 376, 1:1 (M+H)⁺. Suzuki reactionwith phenyl boronic as in Example 1 Step 2 gave6-([1,1′-biphenyl]-4-ylmethyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-onein 69% yield after purification by flash chromatography: LCMS 372.2(M+H)⁺. LDA (0.5 M, 0.6 mL, 0.3 mmol), was added to a solution of theprevious product (108 mg, 0.29 mmol) in THF at −78° C. The reactionmixture was stirred for 30 min. at the same temperature followed byaddition of a solution of iodine (74 mg, 0.29 mmol) solution in THF. Thereaction mixture was allowed to stir for additional 20 min at −78° C.then warmed up to room temperature. After 2h, the reaction was quenchedby sat. aq. NH₄Cl solution and diluted with ethyl acetate. The aqueousphase was extracted with EtOAc (10 mL×2) and the combined organic layerswere washed with brine, dried over Na₂SO₄, filtered and concentrated.The crude product was purified by flash chromatography to afford6-([1,1′-biphenyl]-4-ylmethyl)-7-iodo-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one(80 mg, 55%) as a off white solid. LCMS 498.2 (M+H)⁺.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 to give the title product, which was purified by flashchromatography and isolated in 36% yield as a white solid: LCMS 496.2(M+H)⁺.

Example 426-([1,1′-biphenyl]-4-ylmethyl)-7-(3-(1-hydroxyethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: see Example 13.

Step 2: The Suzuki reaction followed the general procedure from Example1 step 2 using the iodide as described in Example 41 to give the titleproduct, which was purified by flash chromatography and isolated in 37%yield as a white solid: LCMS 492.2 (M+H)⁺.

General Synthesis Scheme for Examples 43-45:

Example 436-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: To a stirred solution of ethyl3,5-dimethyl-1H-pyrazole-4-carboxylate (500 mg, 3.51 mmol) indichlormethane (7 mL), was added triethylamine (0.98 mL, 7.02 mmol) andp-tosyl chloride (1.02 g, 5.35 mmol) at room temperature. The reactionwas allowed to stir for 1h and then water (10 mL) was added and thesolution was extracted with dichlormethane (30 mL×2). The combinedorganic extracts were washed with brine, dried over Na₂SO₄, concentratedunder vacuum and the residue was purified by flash chromatography onsilica gel using 12% of ethyl acetate in hexanes to afford the desiredcompound ethyl 3,5-dimethyl-1-tosyl-1H-pyrazole-4-carboxylate (620 mg,55%). MS (ES) m/z: 323.1 (M+H)+. This compound was noted to be sensitiveto decomposition upon storage and was used immediately in the next step.

Step 2: To a stirred solution of ethyl3,5-dimethyl-1-tosyl-1H-pyrazole-4-carboxylate (500 mg, 1.55 mmol) indry THF was added a solution of LiAlH₄ in THF (2.3 mL, 2.32 mmol) at 0°C. The reaction mixture was allowed to stir for 30 min and then quenchedby dropwise addition of ethyl acetate followed by water. The reactionmixture was partitioned between ethyl acetate and water. The combinedorganic extracts were washed with water and brine and dried over Na₂SO₄.Removal of solvents under vacuum afforded the desired compound(3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methanol (400 mg, 92%). ¹H NMR(CDCl₃, 400 MHz) 7.88-7.85 (m, 2H), 7.34-7.32 (m, 2H), 4.45 (s, 2H),2.53 (s, 3H), 2.44 (s, 3H), 2.29 (s, 3H).

Step 3: To a stirred solution of(3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methanol (76 mg, 0.27 mmol),4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one (50 mg,0.24 mmol), and triphenylphosphine (71 mg, 0.27 mmol) in 1,2dichloroethane (4 mL) under argon was added carbon tetrabromide (89 mg,0.27 mmol) at room temperature. The reaction was allowed to stirovernight. The reaction was quenched by the addition of water (10 mL).Extraction with ethyl acetate (20 mL×2), washing with water and brine,drying with Na₂SO₄, and concentration under vacuum gave a crude residuethat was purified by flash chromatography on silica gel using 70% ethylacetate in hexanes to afford the desired compound6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one(75 mg, 66%), MS (ES) m/z: 468.1 (M+H)+.

Step 4: LDA (0.5 M, 0.3 mL, 0.15 mmol), was added to a solution of6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one(68 mg, 0.145 mmol) in THF at −78° C. The reaction mixture was stirredfor 30 min. at the same temperature followed by addition of a solutionof bispinacolatodiboron (51 mg, 0.2 mmol) solution in THF. The reactionmixture was allowed to stir for additional 20 min at −78° C. then warmedup to room temperature. After 2h, the reaction was quenched by. aq.NH₄Cl solution. This solution was stirred for 1 h and then diluted withethyl acetate. The aqueous phase was extracted with ethyl acetate (10mL×2) and the combined organic layers were washed with brine, dried overNa₂SO₄, filtered and concentrated. The crude product was purified byflash chromatography to afford 21 mg (28%) of(6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-4-isobutyl-2-methyl-1-oxo-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-7-yl)boronicacid: MS (ES) m/z: 512.2 (M+H)+.

Step 5 followed the general Suzuki reaction protocol of Example 1 step2, using (3-(hydroxymethyl)phenyl)boronic acid.6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-onewas isolated in 44% yield: MS (ES) m/z: 574.2 (M+H)⁺.

Step 6: To a solution of the N-tosyl product of step 5 (9.2 mg, 0.016mmol) in 2 mL of methanol was added 2 mL of 5N sodium hydroxide solutionat room temperature. After 3h the solution was diluted with water (10mL) and extracted with ethyl acetate (3×10 mL). The organic extractswere washed with brine, dried over Na₂SO₄, filtered and concentrated.The crude product was purified by flash chromatography to afford 5.1 mg(76%) of the title compound: MS (ES) m/z: 420.2 (M+H)⁺.

Example 446-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-7-(2-fluoro-5-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Steps 1-4: see Example 43.

Step 5 followed the general Suzuki reaction protocol of Example 1 step2, using the boronic ester from Example 23 Step 1.6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-onewas isolated in 44% yield: MS (ES) m/z: 574.2 (M+H)+.

Step 6 was performed as described in Example 43. The crude product waspurified by flash chromatography to give the title compound in 58%yield: MS (ES) m/z: 438.2 (M+H)⁺.

Example 456-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-4-isobutyl-2-methyl-7-(3-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Steps 1-4 were shown in Example 43.

Step 5 followed the general Suzuki reaction protocol of Example 1 step2, using the boronic acid prepared in Step 1 of Example 40.6-((3,5-dimethyl-1-tosyl-1H-pyrazol-4-yl)methyl)-4-isobutyl-2-methyl-7-(3-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-onewas isolated in 33% yield: MS (ES) m/z: 642.2 (M+H)

Step 6 was performed as shown in Example 43. Data for the final product:¹H NMR (CDCl₃, 400 MHz) 7.56-7.51 (m, 2H), 7.49-7.43 (m, 3H), 5.26-5.20(m, 1H), 5.08 (s, 2H), 3.49 (s, 3H), 2.16-2.11 (m, 2H), 2.09-2.06 (m,1H), 1.77 (s, 6H), 0.93 (d, 6H, J=6.8). LCMS 488.15 (M+H)⁺.

General Synthesis Scheme for Examples 46-48:

Example 466-((2-chloroquinolin-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: To a dry 100 mL round bottom flask under argon was addedanhydrous DMF (40 mL) and NaH (0.293 g, 60%, 7.31 mmol) then thesolution was cooled to 0° C. In a separate flask4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one (1.021g, 4.87 mmol) was dissolved in DMF (10 mL) and then this solution wasadded dropwise by cannula to the NaH/DMF mixture. The reaction mixturewas stirred at 0° C. for 2 h and then 2-(trimethylsilyl)ethoxymethylchloride (1.067 g, 6.33 mmol) was added. After 10 h saturated NH₄Clsolution (10 mL) was added and this mixture was extracted with ethylacetate. The combined organic layers were washed with water (3×25 mL),brine (1×25 mL), dried over Na₂SO₄ and then concentrated to give a crudesolid which was purified using flash chromatography (hexanes:ethylacetate, 1:1) to give4-isobutyl-2-methyl-6-((2-(trimethylsilyl)ethoxy)methyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-oneas a light brown solid (1.32 g, 82%): ¹H NMR (CDCl₃, 400 MHz) δ 7.58 (d,J=2.0 Hz, 1H), 7.13 (d, J=2.0 Hz, 1H), 5.41 (s, 2H), 3.73 (s, 3H), 3.48(br t, 2H), 2.58 (d, J=7.0 Hz, 2H), 2.20-2.11 (septet, 1H), 0.97 ppm (d,J=6.6 Hz, 6H), 0.92 (br t, 2H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 159.8,145.6, 122.6, 120.6, 118.1, 116.3, 81.2, 68.4, 43.8, 39.3, 29.4, 24.1,19.1, 0.0 ppm; FT-IR (neat, cm⁻¹) 3095.9, 2951.7, 2866.8, 1636.4,1586.7, 1534.7, 1459.8, 1427.8, 1399.8, 1355.7, 1335.8, 1288.9, 1249.6,1203.7, 1167.8, 1146.7, 1093.4, 1072.6, 1036.8, 1016.8, 997.8, 943.7,928.7, 943.7, 928.7, 859.5, 833.4, 789.7, 748.6, 710.7, 697.6 cm⁻¹; HRMS(ES-TOF) m/z: [M+H]+ Calc'd for C₁₇H₂₉N₃O₂Si: 336.2101, Found: 336.2107.

Step 2: To a clean dry flask with stir bar under argon was added theproduct of Step 1 (1 equiv.) and I₂ (2 equiv.) in anhydrous THF. Thereaction mixture was cooled to −78° C. and LDA (0.5 M solution in THF, 3equiv.) was added dropwise over 10 min. The mixture kept at −78° C. for4 h and then was allowed to warm to room temperature over 15 h. MeOH (10mL) was added and the mixture was stirred for 30 min. Saturated NH₄Clsolution (30 mL) was added and the mixture was extracted with ethylacetate. The combined organic layers were washed with water (3×25 mL),brine (1×25 mL), dried over Na₂SO₄ and concentrated to give a crudesolid which was purified using flash chromatography to give the desirediodinated product in 68% yield. ¹H NMR (CDCl₃, 400 MHz) δ 7.42 (s, 1H),5.47 (s, 2H), 3.69 (s, 3H), 3.54 (br t, 2H), 2.53 (d, J=7.0 Hz, 2H),2.16-2.06 (septet, 1H), 0.97 ppm (d, J=6.6 Hz, 6H), 0.92 (br t, 2H) ppm;¹³C NMR (CDCl₃, 100 MHz) δ 159.8, 145.6, 122.6, 120.6, 118.1, 116.3,81.2, 68.4, 43.8, 39.3, 29.4, 24.1, 19.1, 0.0 ppm; FT-IR (neat, cm⁻¹)3095.9, 2951.7, 2866.8, 1636.4, 1586.7, 1534.7, 1459.8, 1427.8, 1399.8,1355.7, 1335.8, 1288.9, 1249.6, 1203.7, 1167.8, 1146.7, 1093.4, 1072.6,1036.8, 1016.8, 997.8, 943.7, 928.7, 943.7, 928.7, 859.5, 833.4, 789.7,748.6, 710.7, 697.6 cm⁻¹; HRMS (ES-TOF) m/z: [M+H]⁺ Calc'd for C₁₇H₂₈1N₃O₂Si: 462.1072, Found: 462.1074.

Step 3 followed the general Suzuki reaction protocol of Example 1 step2, using (3-(methoxycarbonyl)phenyl)boronic acid and the product of Step2 as coupling partners. methyl3-(4-isobutyl-2-methyl-1-oxo-6-((2-(trimethylsilyl)ethoxy)methyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-7-yl)benzoatewas isolated in 67% yield: ¹H NMR (CDCl₃, 400 MHz) δ 8.14 (s, 1H), 8.47(d, J=7.4 Hz, 1H), 8.30 (d, J=7.7 Hz, 1H) 7.64 (t, J=7.6 Hz, 1H), 5.30(s, 2H), 3.92 (s, 3H), 3.68 (s, 3H), 3.43 (obscured t, 2H), 2.24-2.16(septet, 1H), 1.01 (d, J=6.6 Hz, 6H), 0.87 (obscured t, 2H), −0.03 (s,9H); LCMS (ES) m/z: 470.2 (M+H)⁺.

Step 4: To a 100 mL round bottom flask was added the product from step 3(1 equiv) dissolved in DMF. To this was added tetramethyl ethylenediamine (3 equiv) and TBAF (3 equiv). The vessel was fitted with areflux condenser and heated at 45° C. for 20 h. After confirming thecompletion of the reaction by LCMS (m/z 340), the reaction mixture wasallowed to cool to room temperature and to it was added sat. solution ofNH₄Cl. The contents were transferred to a separatory funnel containingwater. Extraction was done using ethyl acetate and the combined organiclayer was washed with water followed by brine, dried over sodiumsulfate, and evaporated to give a crude solid in 98% yield: ¹H NMR(CDCl₃, 400 MHz) δ 9.84 (br s, 1H), 8.14 (s, 1H), 8.40 (d, J=8.0 Hz,1H), 8.34 (s, 1H), 7.95 (d, J=7.8 Hz, 1H) 7.48 (t, J=7.6 Hz, 1H), 7.15(d, J=2.7 Hz, 1H), 3.90 (s, 3H), 3.74 (s, 3H), 2.60 (d, J=7.4 Hz, 2H),2.23-2.12 (septet, 1H), 1.01 (d, J=6.6 Hz, 6H).

Step 5: To the product from step 4 (1 equiv) dissolved in a 1:1 mixtureof toluene: THF was added diisobutylaluminum hydride (1.0 M in hexanes;2 equiv) and the mixture was allowed to stir for 3 h. After confirmingthe completion of the reaction by LCMS (m/z—312), the reaction wasquenched by addition of 1N HCl and filtered to remove the insolublesolids. The contents were transferred to a separatory funnel andextracted using DCM, washed with water and brine, and the organic layerwas dried over sodium sulfate and evaporated to give a crude solid in97% yield: ¹H NMR (CD₃OD, 400 MHz) δ 7.93 (br s, 1H), 7.84 (d, J=7.4 Hz,1H), 7.46-7.43 (m, 2H), 7.40 (br s, 1H), 4.65 (s, 2H), 3.70 (s, 3H),2.66 (d, J=7.4 Hz, 2H), 2.27-2.17 (septet, 1H), 1.01 (d, J=6.6 Hz, 6H)ppm.

Step 6: The alcohol from step 5 was added to a round bottom flask anddissolved in DCM. To this was added TBDMS-Cl (2 equiv) and imidazole (2equiv) and the mixture was allowed to stir for 12 h. After confirmingthe completion of the reaction by LCMS (m/z—426), the solvents wereevaporated and the crude oil was purified by flash chromatography (1:1Hexanes:EtOAc) to give TBDMS protected alcohol as a pure white solid in96% yield: ¹H NMR (CDCl₃, 400 MHz) δ 9.44 (s, 1H), 7.87 (d, J=7.4 Hz,1H), 7.81 (br s, 1H), 7.44-7.40 (obscured t, 1H), 7.350 (br d, 1H), 7.14(d, J=2.8 hZ, 1H), 4.80 (s, 2H), 3.74 (s, 3H), 2.61 (d, J=7.4 Hz, 2H),2.24-2.14 (septet, 1H), 1.01 (d, J=6.6 Hz, 6H), 0.95 (s, 9H), 0.12 (s,6H) ppm.

Step 7: To a dry 100 mL flask with stir bar under argon was addedanhydrous degassed DMF, NaH (1.2 equiv) and TBDMS protected alcohol fromstep 6 (1 equiv). The mixture was allowed to stir for 2 h.Bromomethyl-2-chloroquinoline (1.1 equiv) was dissolved in minimalanhydrous DMF was then added dropwise to the reaction mixture, which wasstirred overnight. At this time LCMS (m/z—602) and TLC analysisindicated full conversion. Addition of NH₄Cl solution, extraction withethyl acetate, washing with water and brine, drying over Na₂SO₄,filtration, and concentration gave a crude solid that was purified byflash chromatography to give alkylated product as a buff solid in 82%yield based on recovered starting material: ¹H NMR (CDCl₃, 400 MHz) δ8.08 (d, J=8.3 Hz, 1H), 7.78 (obscured t, 2H), 7.69 (br d, 2H), 7.57(obscured t, 2H), 7.38-7.35 (m, 5H), 7.06 (s, 1H), 6.67 (s, 1H), 5.68(s, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 2.60 (d, J=7.4 Hz, 2H), 2.19-2.12(m, 1H), 0.99 (d, J=6.6H, 6H), 0.85 (s, 12H), 0.0006 (s, 6H).

Step 8: The alkylated product obtained in step 7 (1 equiv) was dissolvedin THF and to it was added TBAF (1.0 M in THF, 2.0 equiv). Afterstirring for 2 h and confirming the deprotection of the TBDMS group, thesolvents were evaporated and the residue was purified using preparativeHPLC. The fractions corresponding to desired product were collected,concentrated, and freeze-dried to give the final product of this Exampleas a white solid in 65% yield. ¹H NMR (CDCl₃, 400 MHz) δ 8.08 (d, J=7.9Hz, 1H), 7.78 (t, J=8.3 Hz, 1H), 7.69 (br d, 1H), 7.57 (t, J=8.3 Hz,2H), 7.49 (br s, 1H), 7.37-7.33 (m, 2H), 7.08 (s, 1H), 6.67 (s, 1H),5.68 (s, 2H), 4.67 (s, 2H), 3.71 (s, 3H), 2.60 (d, J=7.4 Hz, 2H),2.20-2.10 (m, 1H), 0.99 (d, J=6.6H, 6H).

Example 476-((1-chloroisoquinolin-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Steps 1-6: see Example 46.

Step 7: To a dry 100 mL flask with stir bar under argon was addedanhydrous DMF, NaH (1.2 equiv) and the product from step 6 (1 equiv).The mixture was allowed to stir for 2 h. Bromomethyl1-chloroisoquinoline (1.1 equiv) dissolved in minimal anhydrous DMF wasthen added dropwise to the reaction mixture, which was stirred overnightwith warming to room temperature. At this time LCMS (m/z—602) and TLCanalysis indicated full conversion. Addition of NH₄Cl solution,extraction with ethyl acetate, washing with water and brine, drying overNa₂SO₄, filtration, and concentration gave a crude solid that waspurified using flash chromatography to give a buff solid in 71% yield:¹H NMR (CDCl₃, 400 MHz) δ 8.41 (obscured d, 1H), 7.98 (s, 1H), 7.70 (brd, 2H), 7.48-7.46 (m, 4H), 6.87 (s, 1H), 5.56 (s, 2H), 4.76 (s, 2H),3.67 (s, 3H), 2.46 (d, J=7.4 Hz, 2H), 2.09-1.99 (septet, 1H), 0.91 (s,3H), 0.89 (s, 12H), 0.06 (s, 6H).

Step 8 followed the procedure of Step 8 in Example 46, giving the titledproduct as a white solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.41 (obscured d,1H), 7.98 (s, 1H), 7.70 (br d, 2H), 7.48-7.46 (m, 5H), 6.87 (s, 1H),5.56 (s, 2H), 4.76 (s, 2H), 3.67 (s, 3H), 2.46 (d, J=7.4 Hz, 2H),2.09-1.99 (septet, 1H), 0.89 (d, J=6.6 Hz, 6H)

Example 486-(2-(2-chlorophenoxy)ethyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Steps 1-6: see Example 46.

Step 7-8: To a dry 50 mL flask with stir bar under argon and cooled to0° C. was added anhydrous DMF, NaH (1.2 equiv), the product from step 6of example 46 (1 equiv) and the mixture was allowed to stir for 2 h.1-(2-bromoethoxy)-2-chlorobenzene (Fort et. al. Synthesis 2004, 15,2527) (1.5 equiv) was dissolved in minimal DMF was then added dropwiseto the reaction mixture, which was stirred overnight with warming toroom temperature. At this time LCMS (m/z—581) and TLC analysis indicatedfull conversion. Addition of NH₄Cl solution, extraction with ethylacetate, washing with water and brine, drying over Na₂SO₄, filtration,and concentration gave a crude solid (˜25 mg) that was re-dissolved inTBAF solution (1.0 M in THF, 2.0 equiv). After stirring for 2 h andconfirming the deprotection of the TBDMS group, the solvents wereevaporated and the residue was purified using preparative HPLC. Thefractions corresponding to desired product were collected, concentrated,and freeze-dried to give the final product of this Example as a whitesolid in 40% yield over two steps: ¹H NMR (CD₃OD, 400 MHz) δ 7.55 (s,1H), 7.27-7.22 (br, 4H), 7.14 (br d, 1H), 6.99 (br t, 1H), 6.71(obscured t, 2H), 4.46 (s, 1H), 4.33 (br t, 2H), 4.06 (br t, 2H), 3.41(s, 3H), 2.43 (d, J=7.4 Hz, 2H), 2.04-2.00 (m, 1H), 0.78 (d, J=6.6 Hz,6H).

Synthesis Scheme for Examples 49-52:

Example 496-((2-((5-aminopentyl)amino)quinolin-4-yl)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1 To a microwave vial containing product from step 7 from example46 (1 equiv) added NaOtBu (1.5 equiv), Pd(OAc)₂ (0.001 equiv), BINAP(0.002 equiv), 1,5-diamino pentane (3 equiv) and toluene (5 mL). Thecontents were mixed well and then degassed by bubbling with argon for 5min. The reaction mixture was then subjected to microwave heating for 3h at 100° C. After confirming the completion of the reaction by LCMS(m/z 667), the reaction mixture was filtered to remove insoluble solidsand the evaporation of solvents give the coupling product as a crude oilthat was taken to the next step.

Step 2: The crude from step 1 was dissolved in THF and to it was addedTBAF (1.0 M in THF, 2.0 equiv). After stirring for 2 h and confirmingthe deprotection of TBDMS group by LCMS, the solvents were evaporatedand the residue was purified using preparative HPLC. The fractionscorresponding to desired product were collected, concentrated, andfreeze-dried to give the final product of this Example as a white solidin 45% yield: ¹H NMR (CD₃OD, 400 MHz) δ 7.91 (s, 1H), 7.79 (br d, 1H),7.56 (br s, 2H), 7.50-7.46 (m, 1H), 7.47-7.43 (m, 3H), 7.15 (s, 1H),6.85 (s, 1H), 5.48 (br t, 1H), 5.34 (s, 2H), 4.82 (s, 2H) 4.76 (s, 2H),3.67 (s, 3H), 3.65-3.62 (obscured t, 2H), 2.75 (br t, 2H), 2.41 (d,J=7.4 Hz, 2H), 2.02-1.97 (m, 1H), 1.56 (br m, 6H), 0.93 (s, 9H), 0.86(d, J=6.6 Hz, 6H), 0.10 (s, 6H).

Example 506-((1-((5-aminopentyl)amino)isoquinolin-411)methyl)-7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one

Step 1: Followed Step 1 of Example 49 using the product from step 7 fromexample 47.

Step 2: Followed Step 2 of Example 49: ¹H NMR (CDCl₃, 400 MHz) δ 7.91(s, 1H), 7.79 (br d, 1H), 7.56 (br s, 2H), 7.50-7.46 (m, 1H), 7.47-7.43(m, 3H), 7.15 (s, 1H), 6.85 (s, 1H), 5.48 (br t, 1H), 5.34 (s, 2H), 4.82(s, 2H) 4.76 (s, 2H), 3.67 (s, 3H), 3.65-3.62 (obscured t, 2H), 2.75 (brt, 2H), 2.41 (d, J=7.4 Hz, 2H), 2.02-1.97 (m, 1H), 1.56 (br m, 6H), 0.93(s, 9H), 0.86 (d, J=6.6 Hz, 6H), 0.10 (s, 6H).

Example 51N-(5-((4-((7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-1-oxo-1H-pyrrolo[3,4-d]pyridazin-6(2H)-yl)methyl)quinolin-2-yl)amino)pentyl)acetamide

The product of Step 1 of example 50 was dissolved in dichloromethane.Hunig's base (2 equiv) and acetyl chloride (1.5 equiv) were added andthe reaction mixture was allowed to stir overnight at room temperature.The solvents were evaporated to give a crude oil which was dissolved inTHF and to this was added (n-Bu)₄NF (1.0 M in THF, 2.0 equiv). Afterstirring for 2h the solvents were evaporated and the residue waspurified using preparative HPLC. The fractions corresponding to desiredproduct were collected, concentrated, and freeze-dried to give the finalproduct of this Example as a white solid in 30% yield: ¹H NMR (CD₃OD,400 MHz) δ 8.45 (d, J=8.4 Hz, 1H), 7.89-7.85 (obscured t, 1 H),7.77-7.76 (obscured t, 1H), 7.65 (d, J=8.2 Hz, 1H), 7.63 (s, 1H),7.42-7.38 (br m, 3H), 7.33-7.30 (m, 1H), 6.90 (s, 1H), 5.64 (s, 2H),4.60 (s, 2H), 3.61 (s, 3H), 3.56 (t, J=7.3 Hz, 2H), 3.26 (s, 1H),3.24-3.20 (br t, 2H), 3.11 (s, 1H), 2.60 (d, J=7.4 Hz, 2H), 2.19-2.13(m, 2H), 1.86-1.82 (m, 2H), 1.76-1.68 (m, 2H), 1.56-1.50 (m, 2H), 1.36(s, 1H), 0.95 (d, J=6.6 Hz, 6H).

Example 52(S)-4-amino-6-((5-((4-((7-(3-(hydroxymethyl)phenyl)-4-isobutyl-2-methyl-1-oxo-1H-pyrrolo[3,4-d]pyridazin-6(2H)-yl)methyl)isoquinolin-1-yl)amino)pentyl)amino)-5-oxohexanamide

The product from step 1 from example 50 (1 equiv) was dissolved in DMFand to it was added Boc-OH-glutamine (1.5 equiv), diisopropylethyl amine(2 equiv), and HATU (1.5 equiv). This mixture was allowed to stirovernight. After confirming the completion of the reaction by LCMS (m/z895) the reaction was quenched by the addition of saturated NH₄Cl andthen extracted with ethyl acetate. After washing the organic layer withwater and brine it was dried over Na₂SO₄, filtered and the solvent wasevaporated to give a crude oil. The crude material was re-dissolved inminimal THF and to it was added excess HCl (4.0 M in 1,4-dioxane). Thisreaction mixture was allowed to stir for 2 h and constantly monitoredfor completion using LCMS (m/z 681). Once the disappearance of startingmaterial was confirmed, the reaction mixture was quenched with NaHCO₃,the organic layer was concentrated and the crude material was purifiedusing preparative HPLC. The fractions corresponding to desired productwere collected, concentrated, and freeze-dried to give the final productof this Example as a white solid in 45% yield: ¹H NMR (CD₃OD, 400 MHz) δ8.45 (d, J=8.4 Hz, 1H), 7.89-7.85 (br t, 1 H), 7.78-7.74 (br t, 1H),7.64 (obscured d, 1H), 7.63 (s, 1H), 7.41-7.39 (br m, 2H), 7.33-7.31 (m,1H), 6.89 (s, 1H), 5.64 (s, 2H), 4.61 (s, 2H), 3.88 (t, 1H), 3.61 (s,3H), 3.56 (t, J=7.3 Hz, 2H), 2.60 (d, J=7.4 Hz, 2H), 2.43-2.38 (m, 2H),2.19-2.03 (m, 4H), 1.86-1.79 (m, 3H), 1.68-1.63 (m, 3H), 1.55-1.49 (m,2H), 1.28 (s, 2H), 0.95 (d, J=6.6 Hz, 6H).

Biological Activity

Example 53 Biological Activity of Selected Compounds of the Invention

Specific Examples 1-52 of compounds of the invention, with estimatedEC₅₀ values determined using an MTT assay for 4-day viability of Raji(Burkitt's) lymphoma cells, a cell line known to highly express MCT1 andto be sensitive to small molecule MCT inhibitors,⁴ are shown in Table 2.Assay protocols follow those described in the literature.⁴ Other assaysthat are not described here but that are standard in the field, such asan assay for competitive inhibition of transport of radiolabeled lacticacid, an MCT substrate, may also be useful in establishing mechanism ofaction of these compounds.

TABLE 2 approximate potency Example (EC₅₀) 1 ≤20 nM 2 100 nM 3 1-10 μM 4200 nM 5 160 nM 6 ≤20 nM 7 ≤20 nM 8 34 nM 9 ≤20 nM 10 90 nM 11 25 nM 1275 nM 13 50 nM 14 <20 nM 15 <20 nM 16 220 nM 17 100 nM 18 900 nM 19 70nM 20 ≤20 nM 21 ≤20 nM 22 ≤20 nM 23 ≤20 nM 24 1 μM 25 300 nM 26 90 nM 2780 nM 28 40 nM 29 80 nM 30 600 nM 31 100 nM 32 900 nM 33 70 nM 34 1 μM35 80 nM 36 100 nM 37 100 nM 38 ≤20 nM 39 90 nM 40 50 nM 41 800 nM 421-10 μM 43 500 nM 44 200 nM 45 ≤20 nM 46 1-10 μM 47 1-10 μM 48 1-10 μM49 1-10 μM 50 ≤20 nM 51 ≤20 nM 52 ≤20 nM

Example 53 Mouse Xenograft Studies

The in vivo effects of a few selected agents have been evaluated inmouse xenograft models and many potent compounds from Example 53 werefound to be effective in arresting tumor growth or in provoking tumorregression.

Protocols follow those described in the literature.⁴ Mice weretransplanted with cultured tumor cells and, after an incubation period(typically 10 days), mice were left untreated or were treated daily witha 30 mg/kg dose of the test compound. Tumor volumes were measured withcalipers over ˜20 days of treatment. Tumors were excised and weighed atthe end of treatment. In two experiments using Raji Burkitt's lymphomacells⁴, the compounds studied were the product of example 9 (FIG. 3),code named SR-11105, and the product of example 21 (FIG. 4), code namedSR-13779.

Treatment with SR-11105 led to a significant reduction of tumor growthrate over time (FIG. 5).

Treatment with SR-13779 effectively blocked tumor growth over time (FIG.6).

A significant reduction in final tumor mass was observed, both whenusing SR-11105 and SR-13779, with tumor mass measured at day 29 in eachcase (FIG. 7). SR-13779 had the more pronounced effect.

Mice that had been treated with either SR-11105 or SR-13779 maintainedtheir body weight over the course of the study (FIG. 8), indicatingthese compounds are safe in treated mice.

Variations

It is understood that certain claimed molecules may stably exist in withisotopic variants among specific substituents, such as deuterium ortritium in the place of hydrogen. Such isotopic variants also fallwithin the scope of the invention.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

It is also understood that certain groups such as amines bear a netcharge. When such a group or groups are present in a “claimed compound”,pharmaceutically acceptable salt forms of the structure are implicitlyencompassed in the claims as well. For example, a claim for a compoundwith one or more amino groups present in the structure also implicitlyclaims all pharmaceutically acceptable salt forms, such ashydrochloride, methanesulfonyl, formate, oxalate, tartrate salts, andthe like.

It is understood that certain “claimed compounds” may stably exist ashydrates or solvates. Such differing forms are also implicitlyencompassed in the claims. Hydrates refer to molecules of water presentin the crystal lattice. Solvates refer to molecules of a relativelybenign solvent, such as ethanol, present in the crystal lattice.

It is understood that certain “claimed compounds” in any form, includingas a salt, hydrate, or solvate, may stably exist in multiple solidcrystalline and/or amorphous forms. Such forms may confer differentphysical properties (e.g., rate of dissolution, stability,hydroscopicity). Such differing solid forms are also implicitlyencompassed in the claims.

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While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements will be apparent to thoseskilled in the art without departing from the spirit and scope of theclaims.

All patents and publications referred to herein are incorporated byreference herein to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. A method of inhibiting monocarboxylatetransporter MCT1, monocarboxylate transporter MCT4, or both, comprisingcontacting the monocarboxylate transporter with an effective amount orconcentration of a compound of formula A

wherein: R¹ is selected from the group consisting of hydrogen,(C₁-C₆)alkyl, (C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, and(C₁-C₆)fluoroalkyl; R² is selected from the group consisting ofhydrogen, (C₁-C₆)alkyl, (C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl,(C₁-C₆)fluoroalkyl, a (C₆-C₁₀)aryl ring system, a 5- to 9-memberedheteroaryl ring system, a (C₁-C₆)alkyl-(C₆-C₁₀)aryl ring system, and a(C₁-C₆)alkyl-(5- to 9-membered)heteroaryl ring system; provided thatwhen R² comprises an aryl or heteroaryl ring system, the ring systembears 0-2 independently selected substituents from the group consistingof fluoro, chloro, trifluoromethyl, (C₁-C₆)alkoxy, and(C₁-C₆)fluoroalkoxy; R³ is a monocyclic or bicyclic (C6-C10) aryl or amonocyclic or bicyclic (5- to 10-membered) heteroaryl group, wherein thearyl or heteroaryl can be substituted or unsubstituted; R⁴ is hydrogen,(C₁-C₆)alkyl, (C₃-C₆)branched alkyl, (C₃-C₇)cycloalkyl, (C₆-C₁₀)aryl,(5- to 7-membered)heteroaryl, or (4- to 7-membered) saturatedheterocyclyl with 1-2 instances of heteroatoms selected from the groupconsisting of NH, NMe, O, and S; Z is CH₂, CH((C₁-C₆)alkyl),CH((C₃-C₇)cycloalkyl), O, N, S, S(O), or SO₂; n=1, 2, or 3; the cyclicgroup indicated as “ring” is an aryl or heteroaryl group of any one ofthe following:

wherein wavy lines indicate points of bonding, and wherein M isindependently selected CH or N, provided that M group can be a nitrogenatom in 0, 1, or 2, instances; L is S, O, NH, N(C₁-C₆)alkyl, or NCF₃,each Q is independently CH or N; wherein R⁵ is optionally present, whenpresent, R⁵ is one to four instances of independently selected F, Cl,Br, CF₃, (C₁-C₆)alkyl, OCF₃, O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl; or, thecyclic group indicated as “ring” is a (C₃-C₇)cycloalkyl or a saturated(3- to 7-membered)heterocyclyl comprising 1-2 heteroatoms selected fromthe group consisting of O, NH, N(C1-C6)alkyl, and N(C1-C6)fluoroalkyl;wherein the points of bonding may be cis or trans; wherein R⁵ isoptionally present, when present, R⁵ is one to four instances ofindependently selected F, Cl, Br, CF₃, (C₁-C₆)alkyl, OCF₃,O(C₁-C₆)alkyl, or CO—(C₁-C₆)alkyl; or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein for the compound of formula Athe R³ group is monocyclic, and is of one of the following formulas

wherein X is H, (C₁-C₆)alkyl, or CF₃; and Y is optionally present and,when Y is present, Y is 1-3 instances of a substituent selected from thegroup consisting of F, Cl, Br, CF₃, (C₁-C₆)alkyl, O(C₁-C₆)alkyl, NH₂,NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂, NH—(CH₂)_(j)—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding.
 3. The method of claim1, wherein for the compound of formula A the R³ group is bicyclic, andis of one of the following formulas:

wherein the group X is H, (C₁-C₆)alkyl, or CF₃; and Y is optionallypresent and, when Y is present, Y is 1-3 instances of a substituentselected from the group consisting of F, Cl, Br, CF₃, (C₁-C₆)alkyl,O(C₁-C₆)alkyl, NH₂, NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂,NH—(CH₂)_(j)—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding, and wherein Y can bedisposed on any ring of a multi-ring system.
 4. The method of claim 1,wherein for the compound of formula A the R³ group is bicyclic and is ofone of the following formulas:

wherein the group X is H, (C₁-C₆)alkyl, or CF₃; and Y is optionallypresent and, when Y is present, Y is 1-3 instances of a substituentselected from the group consisting of F, Cl, Br, CF₃, (C₁-C₆)alkyl,O(C₁-C₆)alkyl, NH₂, NH(C₁-C₆)alkyl, N((C₁-C₆)alkyl)₂,NH—(CH₂)_(j)—CH₂-Q, and

wherein j=2-6 and Q is one of the following groups

wherein a wavy line indicates a point of bonding, and wherein Y can bedisposed on any ring of a multi-ring system.
 5. The method of claim 1,wherein R¹ is Me or cycloalkyl, R² is alkyl or branched alkyl, Z is CH₂or CH(Me), R⁴ is H, Me, or CF₃, the cyclic group indicated as “ring” isaryl or heteroaryl, and all other groups are as specified in claim
 1. 6.The method of claim 1, wherein the compound of formula A is any one ofthe following, including all stereoisomeric forms, all isotopic forms,all crystalline and amorphous forms, and all pharmaceutically acceptablesalt forms thereof: